Carboxy-terminal domain mediates assembly of the voltage-gated rat ether-a-go-go potassium channel
1997; Springer Nature; Volume: 16; Issue: 21 Linguagem: Inglês
10.1093/emboj/16.21.6337
ISSN1460-2075
Autores Tópico(s)Neuroscience and Neural Engineering
ResumoArticle1 November 1997free access Carboxy-terminal domain mediates assembly of the voltage-gated rat ether-à-go-go potassium channel Jost Ludwig Jost Ludwig Zentrum für Molekulare Neurobiologie der Universität Hamburg, Institut für Neurale Signalverarbeitung, Martinistrasse 52, D-20246 Hamburg, Germany Search for more papers by this author David Owen David Owen Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT UK Search for more papers by this author Olaf Pongs Corresponding Author Olaf Pongs Zentrum für Molekulare Neurobiologie der Universität Hamburg, Institut für Neurale Signalverarbeitung, Martinistrasse 52, D-20246 Hamburg, Germany Search for more papers by this author Jost Ludwig Jost Ludwig Zentrum für Molekulare Neurobiologie der Universität Hamburg, Institut für Neurale Signalverarbeitung, Martinistrasse 52, D-20246 Hamburg, Germany Search for more papers by this author David Owen David Owen Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT UK Search for more papers by this author Olaf Pongs Corresponding Author Olaf Pongs Zentrum für Molekulare Neurobiologie der Universität Hamburg, Institut für Neurale Signalverarbeitung, Martinistrasse 52, D-20246 Hamburg, Germany Search for more papers by this author Author Information Jost Ludwig1, David Owen2 and Olaf Pongs 1 1Zentrum für Molekulare Neurobiologie der Universität Hamburg, Institut für Neurale Signalverarbeitung, Martinistrasse 52, D-20246 Hamburg, Germany 2Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT UK *E-mail: [email protected] The EMBO Journal (1997)16:6337-6345https://doi.org/10.1093/emboj/16.21.6337 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The specific assembly of subunits to oligomers is an important prerequisite for producing functional potassium channels. We have studied the assembly of voltage-gated rat ether-à-go-go (r-eag) potassium channels with two complementary assays. In protein overlay binding experiments it was shown that a 41-amino-acid domain, close to the r-eag subunit carboxy-terminus, is important for r-eag subunit interaction. In an in vitro expression system it was demonstrated that r-eag subunits lacking this assembly domain cannot form functional potassium channels. Also, a ∼10-fold molar excess of the r-eag carboxy-terminus inhibited in co-expression experiments the formation of functional r-eag channels. When the r-eag carboxy-terminal assembly domain had been mutated, the dominant-negative effect of the r-eag carboxy-terminus on r-eag channel expression was abolished. The results demonstrate that a carboxy-terminal assembly domain is essential for functional r-eag potassium channel expression, in contrast to the one of Shaker-related potassium channels, which is directed by an amino-terminal assembly domain. Introduction Voltage-gated potassium (Kv) channels play an important role in controlling resting membrane potential in neuronal as well as in non-neuronal cells. In excitable cells of the nervous system they are not only responsible for repolarization of action potentials, but also for modulating their form and frequency (Hille, 1992); Kv channels are therefore an important factor in neuronal signal transmission and processing. Kv channels in the Shaker family, consist of four identical or homologous α subunits (MacKinnon, 1991; Liman et al., 1992) and may have four additional modulatory β-subunits (Rehm and Lazdunski, 1988; Parcej and Dolly, 1989; Parcej et al., 1992; Rettig et al., 1994). Each α-subunit is characterized by six putative transmembrane segments (S1–S6), with the region that is mainly responsible for forming the ion-conducting pore (H5) located between S5 and S6. The assembly of Kv channel α-subunits is primarily mediated by a region within the intracellular amino-terminus known as the tetramerization domain (T1-domain; Li et al., 1992; Shen et al., 1993; Deal et al., 1994; Hopkins et al., 1994; Shen and Pfaffinger, 1995; Xu et al., 1995). This domain also confers sub-family specificity upon heteromultimeric assembly of K-channels. Furthermore, the same region has also shown to be involved in binding of β-subunits to α-subunits (Sewing et al., 1996; Yu et al., 1996). In the ether-à-go-go (eag) family of channels, the subunits probably share the same structural features as Shaker-type channels (i.e. six putative transmembrane regions per subunit, and an H5 region), but the overall sequence similarity between eag and Shaker-type channels is quite low except for the H5 region. A much higher degree of homology is found between eag-type channels and inwardly rectifying K-channels (AKT1, KAT1) from plants (Anderson et al., 1992; Sentenac et al., 1992) and also between eag-type channels and the non-selective cyclic nucleotide-gated (cng) cation channels. In addition, eag channels have a region of yet unknown function within the C-terminus which is homologous to the cyclic nucleotide-binding domain of cng channels. It is presumed that eag-type channels consist of tetramers as the related cng family of ion channels (Kaupp et al., 1989; Liu et al., 1996). However, in contrast to Shaker-type channels, it is not yet known which protein domains are involved in the assembly of functional cng, AKT or eag channels. In order to address this question, in the present study we have used a member of the eag-family, rat eag (r-eag) to study the interaction between subunits. Our approach combined the complementary techniques of a protein overlay assay and functional expression of r-eag-mediated ionic currents in a heterologous mammalian expression system. Our results provide evidence that, in contrast to Kv channels in which an N-terminal domain (T1) is crucial for assembly, r-eag assembly is mediated by a C-terminal domain. Results r-eag carboxy-terminus suppresses expression of r-eag-mediated current It has been shown that the α-subunits of Shaker-type Kv channels contain within the cytoplasmic amino-terminus a domain required for tetramerization and α-subunit assembly. Co-expression of this domain, i.e. the amino-terminus, with wild-type (wt) Shaker subunits in Xenopus oocytes suppressed the formation of functional Kv channels (Li et al., 1992). Most likely, the amino-terminus interfered with the assembly of wild-type subunits. In a first functional screen for possible assembly domains of r-eag subunits, we tested whether formation of functional r-eag channels could be suppressed by co-injection of wt r-eag mRNA with mRNA encoding the amino- (r-eag1–208) or carboxy-terminal (r-eag650–962) part of the r-eag protein. Microinjection of r-eag mRNA into Chinese hamster ovary (CHO) cells resulted in the expression of a delayed-rectifier type voltage-gated K+ current. Currents were recorded in whole-cell patch–clamp mode between 1 and 14 h after injection of r-eag RNA and on average the peak current amplitudes reached a maximum between 4 and 8 h. Between these times, the current was seen in >90% of the injected cells and at +60 mV averaged 1.24 ± 0.16 nA (n = 18). Typically for r-eag currents, the rate of activation was slowed dramatically from hyperpolarized holding potentials. The properties of r-eag-mediated currents in the CHO expression system were as previously described for r-eag currents when expressed in Xenopus oocytes or in 293 cells (Ludwig et al., 1994; Stansfeld et al., 1996; Terlau et al., 1996). Co-injection of r-eag1–208 with r-eag wt RNA (in a 10:1 molar ratio) led to a small suppression of r-eag currents (current mean at + 60 mV: 0.69 ± 0.25 nA, n = 9) (Figure 1A) that was not statistically significant (P > 0.05). In contrast, the co-injection of r-eag650–962 with r-eag RNA (1:1 molar ratio) resulted in a partial suppression of r-eag current (not shown). When the r-eag650–962 RNA was injected in a 10-fold molar excess, virtually complete suppression of the current was observed (mean current at +60 mV: 0.13 ± 0.04 nA, n = 18) (Figure 1A). To rule out the possibility that the suppression of current is due to a non-specific binding of the r-eag650–962 fragment to membrane proteins or due to saturation of intracellular protein synthesis or trafficking pathways, we used the expression of a distantly related Kv channel (Kv 1.5) as a control. Kv1.5 RNA was co-injected with a 10-fold molar excess of r-eag1–208 or r-eag650–962 RNA. Neither of these significantly reduced Kv1.5 currents (Figure 1B). Mean current amplitudes were 1.21 ± 0.39 nA at +60 mV (n = 3) for Kv1.5 alone compared with 1.33 ± 0.19 nA (n = 5) and 1.07 ± 0.27 nA (n = 6) for Kv1.5 plus r-eag1–208 and r-eag650–962, respectively. Figure 1.Co-expression of r-eag subunits with a carboxy-terminal r-eag fragment suppresses functional expression of r-eag currents. (A) Data pooled from a number of experiments in which mRNAs encoding either r-eag, r-eag plus an amino-terminal fragment (r-eag1–208) or r-eag plus a carboxy-terminal fragment (r-eag650–962) was injected into CHO cells. Currents were recorded after 4 h and peak amplitudes were measured following a 500 ms voltage step to +60 mV. The histogram represents the mean of measurements from between 9 and 18 different cells; vertical bars represent the SEM in each case. The difference between the mean amplitudes of currents recorded following co-injection of r-eag with r-eag650–962 (right) and r-eag mRNA alone (left) is highly significant (P 0.05) (Statistical analysis using Student's t-test). (B) Experiments carried out as in (A) except that Kv1.5 mRNA was injected alone or with r-eag1–208 and r-eag650–962 mRNA. Descriptions of the histogram and vertical bars are as for (A). Means were derived from between three and six cells. There was no statistically significant difference between any of the columns (Student's t-test). Download figure Download PowerPoint Functional r-eag expression abolished by carboxy-terminal deletions The results shown in Figure 1 suggested that the co-expression of r-eag subunits with the carboxy-terminus of r-eag suppressed the expression of r-eag-mediated current. Also, it has been shown that the heterologous expression of an amino-terminally truncated r-eag construct, lacking the first 190 aa residues, produces functional r-eag channels (Terlau et al., 1997). Thus, we hypothesized that the carboxy-terminus of r-eag subunits contains sequences important for the formation of functional r-eag channels. Accordingly, we investigated the ability of carboxy-terminal deletion mutants to form functional r-eag channels in the CHO expression system. Injection of RNA corresponding to r-eag1–937 and r-eag1–915 led to the expression of functional channels with characteristics and mean currents that were similar to wild-type (Figure 2A–C, E). This indicated that the 47 carboxy-terminal amino acid residues of r-eag protein, which were deleted in r-eag1–915, are not essential for functional r-eag expression in CHO cells. However, deletion of the r-eag carboxy-terminus by a further 10 amino acids (r-eag1–905), gave rise to functional channels in only three out of 22 injected cells. The mean of those currents observed was 1.57 ± 0.4 nA at +60 mV which was not significantly different from wild-type currents. No currents were observed following injection of RNA encoding r-eag1–896 (n = 5) and r-eag1–873 (n = 5) (Figure 2D and E). The results indicated that r-eag subunits may contain, in the vicinity of amino acid residue 905, domains(s) critical for the expression of functional r-eag channels in CHO cells. Figure 2.Effect of carboxy-terminal deletions on expression of r-eag currents in CHO cells. Currents measured after injection of mRNA encoding r-eag (A), r-eag1–937 (B), r-eag1–915 (C) and r-eag1–896 (D). In each case, currents were evoked with voltage jumps to 0 mV, +30 mV and +60 mV from a holding potential of −60 mV (traces shown superimposed). (E) Data pooled from a number of cells (3–18) including the constructs illustrated in (A–D) and in addition carboxy–terminal deletions r-eag1–905 and r-eag1–873. * indicates that r-eag1–905 was expressed in only three of 22 cells injected. Download figure Download PowerPoint Characterization of carboxy-terminal assembly domain in r-eag subunits Next, we expressed fusion protein constructs (Figure 3A) of maltose binding protein (Mal) and r-eag (Malr-eag) in Escherichia coli (see Materials and methods). The bacterial lysates containing Malr-eag protein constructs were separated on SDS–PAGE (Figure 3B) and blotted onto nitrocellulose membranes for protein overlay binding assays with 35S-labelled r-eag ([35S]r-eag) (Figure 3C) or [35S]r-eag1–208 (Figure 3D) as probe. [35S]r-eag strongly interacted with Malr-eag, Malr-eag272–962, Malr-eag384–962, Malr-eag482–962 and Malr-eag650–937; weaker binding was observed with Malr-eag650–929; and no interaction was visible with Malr-eag 650–915. The [35S]r-eag probe, which contained only amino-terminal residues 1–208 ([35S]r-eag1–208), did not bind to the Malr-eag fusion proteins (Figure 3D), whereas the complementary probe [35S]r-eag191–962 interacted with the Malr-eag fusion proteins (see Figure 4). The data showed that amino-terminal cytoplasmic domains were not required for r-eag subunit interaction in the overlay binding experiments and that r-eag domain(s) between residues 650 and 937 were sufficient for r-eag subunit interaction. The results of the overlay binding experiments strongly supported our interpretation of the functional r-eag expression studies in Figures 1 and 2. Namely, the carboxy-terminus of r-eag subunits contains in the vicinity of amino acid residues 905 important domain(s) for r-eag subunit assembly. Figure 3.Truncated r-eag proteins bind [35S]r-eag, but not [35S]r-eag amino-terminus. (A) Diagram illustrating fusion protein constructs between maltose binding protein and truncated r-eag proteins (Malr-eag) used in the overlay protein binding assays. Numbers for first and last r-eag residues (aa) in the fusion proteins are given on the right. Full-length r-eag protein (1–962) is shown on top. Black boxes indicate the putative transmembrane regions S1–S6; hatched boxes indicate the region that exhibits high similarity to the cyclic nucleotide binding domain of cyclic nucleotide-gated channels. (B) Coomassie blue (CB) stained SDS–polyacrylamide gel of total bacterial lysates from E.coli expressing the Malr-eag fusion protein constructs shown in (A). Expression of shorter Malr-eag constructs (Malr-eag650–937, Malr-eag650–929, Malr-eag650–915) was more efficient than the one of the longer Malr-eag constructs. Accordingly, the amount of bacterial protein was adjusted to load comparable amounts of Malr-eag fusion protein. The positions of molecular weight markers (kDa) are indicated on the left. Fusion protein bands are marked by arrows. (C and D) The blots were incubated with in vitro-translated 35S-labelled r-eag protein ([35S]r-eag) (C) or r-eag amino-terminus ([35S]r-eag1–208) (D). Bound r-eag protein was visualized by autoradiography. Download figure Download PowerPoint Figure 4.Interaction of carboxy-terminal r-eag fragments with 35S-labelled r-eag191–962 and 35S-labelled cad. (A) Diagram illustrating fusion protein constructs between maltose binding protein and carboxy-terminal r-eag fragments (Malr-eag). Numbers for first and last r-eag residues (aa) in the fusion proteins are given on the right. Full-length r-eag protein is shown on top, as in Figure 3A. Shaded area (residues 897–937) indicates carboxy-terminal r-eag binding region. (B) Coomassie blue-stained polyacrylamide gel (CB) of lysates from E.coli expressing the Malr-eag fusion proteins shown in (A), lane 1 (Malr-eag650–937) corresponds to lane 5 in Figure 3A. The r-eag residues within the constructs are indicated on top of each lane, positions of molecular weight markers (kDa) on the left. Blots of equivalent gels were overlaid with in vitro-translated [35S]r-eag191–962 (C) and [35S]r-eag897–937 (cad) (D). Bound peptides were visualized by autoradiography. [35S]r-eag191–962 and [35S]cad binding results have been evaluated according to the signal intensities obtained in the overlay assays shown in C and D. +++, very strong signal intensity; ±, very weak binding; −, no detectable binding. Evaluations are given next to each Malr-eag fusion protein in (A). Download figure Download PowerPoint To define the approximate amino-terminal border of the r-eag subunit interaction recognition domain(s) more precisely, we generated protein blots with Malr-eag fusion protein constructs in which part of the r-eag carboxy-terminus (residues 650–937) was further truncated from its amino- and carboxy-terminal end (Figure 4A). The blots were probed with [35S]r-eag191–962 (Figure 4B and C). When the carboxy-terminus had progressively been shortened from residue 650 to residue 897, only a gradual and relatively small reduction in the intensity of the binding signal was observed. However, when further amino acids had been deleted, as in the case of the Malr-eag906–937 fusion protein, [35S]r-eag binding was markedly decreased. No [35S]r-eag binding was observed with Malr-eag918–937. Also, [35S]r-eag191–962 binding to Malr-eag650–929 was markedly decreased in comparison with Malr-eag650–937 (Figure 4C). Thus, a relatively small carboxy-terminal domain (residues 897–937) was important for r-eag subunit interaction in the overlay binding assays. In contrast, Shaker-related Kv channels contain an amino-terminal subunit interaction domain (Li et al., 1992; Shen et al., 1993; Shen and Pfaffinger, 1995). Since r-eag subunit interaction apparently differs completely from that of Kv channels, we propose to refer to this domain as cad (Carboxy-terminal Assembly Domain). When we used the cad-sequence ([35S]cad) as probe in the overlay-binding experiments (Figure 4D), [35S]cad and [35S]r-eag binding results to Malr-eag650–937 were similar. However, [35S]cad and [35S]r-eag binding to Malr-eag906–937, Malr-eag918–937 and Malr-eag650–929 showed interesting differences (summarized in Figure 4A). Unlike [35S]r-eag, [35S]cad bound equally well to Malr-eag897–937 and Malr-eag906–937 and bound weakly to Malr-eag918–937 in the overlay binding assays (Figure 4D). Also, in contrast to [35S]r-eag, [35S]cad binding to Malr-eag650–929 was not detectable. Apparently, [35S]cad binding was less sensitive than [35S]r-eag to deletions at the amino-terminal cad side and more sensitive than [35S]r-eag to deletions at the carboxy-terminal cad side. Two subdomains in carboxy-terminal r-eag assembly domain Alignment of the Drosophila (D-eag) (Warmke et al., 1991; Brüggemann et al., 1993) and r-eag cad sequences showed that 12 out of 41 amino acid residues are identical and 18 are conservatively substituted (Figure 5). Since 35S-labelled D-eag probes interacted with the r-eag cad region in overlay binding assays (data not shown), it is most likely that conserved cad amino-acid residues are involved in cad/cad interaction and thus, in r-eag subunit assembly. Some of these amino acid residues were mutated by site-directed in vitro mutagenesis of Malr-eag650–937 expression plasmids. As shown in Figure 5, charged amino acids were replaced by neutral amino acid residues or by residues of opposite charge. Alternatively, hydrophobic amino acids (I, L, V) were substituted with alanine. We did not readily obtain cad single-point mutations, which strongly reduced or diminished [35S]r-eag binding. Therefore, we introduced multiple mutations into cad (Figure 5). Substitution of the four negatively charged amino acid residues E901, E905, E908, D909 and the positively charged lysine K 911 by glutamine (Malr-eag650–937,n901–911) caused a marked reduction in [35S]r-eag binding (Figure 5). Also, the substitution of V902, E905 and L906 by alanine and glutamine, (Malr-eag650–937,VEL/AQA) affected [35S]r-eag binding. The strongest reduction in [35S]r-eag binding was detected when the four hydrophobic residues L900, V902, L906 and I910 had been replaced with alanine (Malr-eag650–937,h900–910). In contrast, mutations, which were introduced in the carboxy-terminal half of cad (E922Q/E926Q; E922K/E926Q; L928A/L931A), did not markedly affect [35S]r-eag binding (Figure 5). Figure 5.Influence of point mutations on r-eag subunit interaction. Alignment of Drosophila (D)-eag (residues 918–948) and rat (r)-eag (residues 897–937) cad sequences is shown. Identical residues are shaded, similar residues are boxed. Point mutations indicated below were introduced into Malr-eag650–962. Naming of mutants is given at left. Mutant fusion proteins were tested in overlay binding assays with [35S]r-eag191–962 (not shown). Binding results (classified as in Figure 4) are shown on the right. Download figure Download PowerPoint The results suggested that cad function was more sensitive to amino-terminal than carboxy-terminal amino acid substitutions. Also, relatively small deletions from both cad ends affected markedly r-eag subunit assembly. These observations suggested to us that cad might be divided into two subdomains, A and B (Figure 6A). The subdomains could bind to each other either in an A–A, B–B or in an A–B manner. To test these alternatives we examined in the overlay assay various cad-mutants, where either the A or the B subdomain was mutated. The mutants Malr-eag650–937,h900–910, Malr-eag650–937,n901–911 and Malr-eag906–937 were taken as A-subdomain mutants (Malr-eag in Figure 6C) and Malr-eag650–929 as B-subdomain mutant (Malr-eag in Figure 6C). Blots of the mutant fusion proteins were probed either with probes containing an intact cad sequence ([35S]r-eag; [35S]cad) ('AB'-probe) or with ones containing a mutated A-subdomain ([35S]r-eagh900–910; [35S]r-eagn901–911; [35S]r-eag906–937) ('B+' probe) or a mutated B-subdomain ([35S]r-eag1–929) ('A+'-probe), respectively. The results of the overlay binding experiments using the different A, B and AB fusion proteins and probes are shown in Figure 6B. The various A- and B-subdomain mutations had distinct effects on cad-binding as summarized schematically in Figure 6C. The data indicate that the 'A+'-probe [35S]r-eag1–929 bound only to the Malr-eag fusion proteins (Malr-eag650–929, Malr-eag650–937, Malr-eag897–937) with an intact A-subdomain and did not bind to the ones with a mutated A-subdomain (Malr-eagh900–910, Malr-eagn901–911, Malr-eag906–937). Similarly, [35S]r-eag 'B+'-probes bound only to the Malr-eag fusion proteins with an intact B-domain (Malr-eagh900–910, Malr-eagn901–911, Malr-eag906–937) and did not bind to the one with a mutated B-domain (Malr-eag 650–929). [35S]r-eag, containing both subdomains A and B, bound to Malr-eag fusion proteins, regardless of whether they carried a mutated subdomain A or B, respectively. Interestingly, binding of [35S]r-eag1–962,h900–910 to the Malr-eag650–937,h900–910 fusion protein was stronger than binding of [35S]r-eag1–962,h900–910 to Malr-eag650–937. The binding data indicated that the cad-subdomains interact with each other in a homophilic A–A, B–B manner (Figure 6C). This suggested that mutations of both cad A- and B-subdomains are necessary to eliminate completely any r-eag subunit interaction. This supposition was tested in the functional CHO expression system. Figure 6.Analysis of cad subdomain interactions. (A) Schematic drawing of cad with arbitrary A- and B- subdomain domains. (B) Coomassie blue-stained polyacrylamide gel of lysates from E.coli expressing Mal fusion proteins with r-eag carboxy-terminus (Malr-eag650–937), r-eag carboxy-terminal point mutations (Malr-eag650–937;h900–910, Malr-eag650–937;n901–911) or deletion mutants (Malr-eag897–937, Malr-eag906–937, Malr-eag650–929) as indicated on top of each lane. Fusion protein bands are marked by arrows. Positions of size markers are given on the left. Blots of equivalent gels were incubated with in vitro-translated 35S-labelled r-eag probes as indicated. Bound [35S]r-eag probes were visualized by autoradiography. (C) Binding results of the overlay assays in (B) have been evaluated according to the signal intensities as in Figure 4. Fusion protein constructs were sorted as A−B+, A+B− and A+B+ as described in the text. Similarly, [35S]r-eag probes were classified as A+, B+ or AB probe, respectively. Shading indicates homotypic A− and B− interactions of cad subdomains. Download figure Download PowerPoint Mutant r-eag carboxy-terminus does not suppress r-eag current Combining the results of the overlay binding assays in Figure 6 and those of r-eag expression in Figure 2 suggested that r-eag1–896 (Figure 2D) did not express functional r-eag channels because the cad-domain had been eliminated. On the other hand, expression of carboxy-terminally truncated r-eag1–915 with a missing cad B-subdomain produced functional r-eag channels (Figures 2C and 7A). Expression of r-eag subunits with a mutated cad A-subdomain (r-eagh900–910) also produced functional r-eag channels (Figure 7B). In contrast, no r-eag currents were detected when we attempted to express r-eag subunits with a mutated cad A- and a missing cad B-subdomain (r-eag1–915,h900–910) (n = 7; Figure 7C). Thus, the cad domain is essential for functional r-eag channel expression (Figure 2). Figure 7.Effect of carboxy-terminal cad mutations on functional r-eag expression in CHO cells. CHO cells were injected with (A) r-eag1–915, (B) r-eagh900–910 and (C) r-eag1–915,h900–910 mRNA ∼6 h before recording using whole-cell patch–clamp method. Conditions as described in Materials and methods. Currents were evoked with voltage jumps to +60 mV from a holding potential of −60 mV. (D) Data pooled from a number of experiments in which mRNAs r-eag, r-eag plus r-eag650–962 or r-eag plus r-eag650–962,h900–910 mRNAs were injected into CHO cells. Currents were recorded after 4 h and peak amplitudes were measured following a 500 ms voltage step to +60 mV. In each case the histogram represents the mean of measurements from between four and 18 different cells, and the vertical bars the SEM. Download figure Download PowerPoint In Figure 1, we showed that the presence of a 10-fold molar excess of r-eag650–962 mRNA suppressed the expression of r-eag channels by r-eag mRNA. By contrast, a 10-fold molar excess of r-eag1–896 mRNA lacking the cad-domain did not abolish the expression of r-eag channels by r-eag mRNA (data not shown). The dominant-negative effect of the r-eag650–692 carboxy-terminus on functional r-eag channel expression was most likely due to an interference with the assembly of r-eag subunits. Accordingly, r-eag carboxy-termini with a mutant cad domain should not be able to interfere with functional r-eag channel expression. Indeed, normal r-eag outward currents were recorded with a mean amplitude at +60 mV of 1.65 ± 0.35 nA (n = 4; Figure 7D) when r-eag mRNA was co-expressed in CHO cells with a 10-fold molar excess of r-eag650–962,h900–910 mRNA,. Similar results were obtained, when r-eag mRNA was co-expressed with a 10-fold molar excess of r-eag650–962,VEL/AQA mRNA (1.19 ± 0.4 nA at +60 mV; n = 3). The results show that cad mutations abolished the dominant-negative effect of the r-eag650–962 carboxy-terminus on functional r-eag channel expression. Thus, the in vitro expression studies with mutant cads confirmed the important role of cad in r-eag subunit assembly. Discussion In the present study we have investigated the subunit assembly requirements of r-eag, a member of the eag family of voltage-gated K-channels (Warmke et al., 1991; Ludwig et al., 1994; Warmke and Ganetzky, 1994) using the complementary methods of protein binding (overlay assay) and functional expression of r-eag channels (mRNA injection into CHO cells and whole-cell patch–clamp). Our results show that assembly of r-eag channels involves a carboxy-terminal domain (cad). The principal evidence for this conclusion is drawn from a number of observations: (i) in protein–protein overlay assays, 35S-labelled r-eag binds to fusion proteins carrying the carboxy-terminus and not to fusion protein carrying the amino-terminus; (ii) injections of subunits truncated from the carboxy-terminus beyond residue 896 do not result in the expression of functional channels; (iii) the carboxy-terminus r-eag (r-eag650–962), but not the amino-terminus of r-eag (r-eag1–208), exerted a dominant-negative effect on r-eag channel expression; and (iv) cad-mutations affect functional r-eag channel expression as well as the dominant-negative effect of co-expressed r-eag carboxy-terminus on r-eag currents. Alternative explanations for the failure of the mRNAs which encoded r-eag mutants to produce functional channels when injected into CHO cells might be: (i) the inability of the cell to transport the protein to the plasma membrane; (ii) improper folding of the peptide chain; and (iii) loss of post-translational modifications necessary for channel function. However, these reasons are unlikely in the case of the dominant-negative effect of the carboxy-terminus since the amino-terminus does not have this effect on r-eag and neither the carboxy- nor amino-terminus has any such effect on the Kv1.5 channel expressed in the same system. Furthermore, the overlay assay gives a direct indication of binding between proteins in a cell-free system and is therefore not subject to
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